Apple crown and collar canker and necrosis caused by Cytospora balanejica sp. nov. in Iran

Apple is the most important fruit tree in West Azarbaijan province of Iran. In a survey of apple orchards, a disease with crown and collar canker and necrosis symptoms was observed in three young apple orchards in Urmia, affecting 15% and 1% of ‘Red Delicious’ and ‘Golden Delicious’ cultivars, respectively. A fungus with typical characteristics of the asexual morph of Cytospora was regularly isolated from the diseased tissues. Morphological characteristics and phylogenetic analyses inferred from the combined dataset of the ITS-rDNA, parts of LSU, tef1-α, rpb2, and act1 genes revealed that the isolates represent a new species of Cytospora, described herein as Cytospora balanejica sp. nov.. The pathogenicity of all isolates was confirmed on apple cv. ‘Red Delicious’ based on Koch’s postulates. Also, the reaction of 12 other apple cultivars was assessed against five selected isolates with the highest virulence. The results showed that except for cv. ‘Braeburn’, which did not produce any symptoms of the disease, the other 11 cultivars showed characteristic disease symptoms including sunken and discolored bark and wood. The mean length of the discolored area was different among the 11 so-called susceptible cultivars, hence cvs. ‘M4’ and ‘Golden Delicious’ showed the highest and the lowest lesion length, respectively. Moreover, the aggressiveness of the five tested isolates was different, and the isolates BA 2-4 and BA 3-1 had the highest and lowest aggressiveness, respectively. Based on our observations on the potential ability of the fungus to cause disease on young and actively growing apple trees, it will be a serious threat to apple cultivation and industry.


Phylogenetic analyses
The phylogenetic analyses of the combined dataset (ITS, LSU, act1, rpb2, and tef 1-a) include 257 Cytospora ingroup strains representing 175 Cytospora species and Diaporthe eres CBS 145040 and Diaporthe vaccinii CBS 160.32 as outgroup strains with a total of 3049 characters (1641 constant sites, 1408 variable sites, 240 parsimonyuninformative sites and 1168 parsimony-informative sites) including gaps (642 for ITS, 574 for LSU, 354 for act1, 743 for rpb2, and 763 for tef1-α) (Table 2).The results of best-fit substitution model evaluation in MrModeltest v2.3 recommended GTR+I+G, GTR+I+G, GTR+I+G, GTR+I+G and HKY+I+G models for ITS, LSU, act1, rpb2 and tef 1-a, respectively (Table 2).The best-scoring RaxML tree with the final ML optimization likelihood value of − 47725.166937(ln) is selected to denote and consider the phylogenetic relationships among the strains (Table 2, Fig. 2).The estimated base frequencies were as follows: A = 0.242358, C = 0.268654, G = 0.259113, T = 0.229874; substitution rates AC = 1.549133,AG = 3.919749, AT = 1.913775,CG = 1.087338,CT = 8.678832, GT = 1.000000; gamma distribution shape parameter α = 0.793946.A summary of phylogenetic information and substitution models for each dataset is provided in Table 2. Topologies of the individual gene trees were determined to be congruent and no conflicts were observed in species delimitation (data not shown).The phylogenetic trees generated from MP (TL = 9556; CI = 0.262; RI = 0.766; HI = 0.738) and BI analyses were topologically similar to the one generated via the ML analysis, and the latter is shown in Fig. 2. Cytospora balanejica represented a monophyletic clade with a high support value (ML/MP/BI = 91/99/1) (marked in pink in Fig. 2).

Pathogenicity trials
Results of pathogenicity tests of the isolates (24 isolates) on shoots of the cv.'Red Delicious' showed sunken discolored lesions around the inoculated sites 14 days post-inoculation.Bark and wood discoloration was extended progressively upward and downward the inoculation site and after 20 days, fungal pycnidia were formed on the discolored bark.Despite this, the mean length of necrotic lesions varied among the isolates and ranged from 45 to 172 mm (Table 1).Also, the results of pathogenicity tests of the most virulent isolate (BA 2-4) under field conditions clearly showed bark and wood discoloration and necrosis 45 days post-inoculation (Fig. 4).Re-isolation of the inoculated fungus and re-identification based on morphological characteristics fulfilled Koch's postulates.All negative controls were asymptomatic and no colonies were obtained from samples taken from the controls.The reaction of 12 tested cultivars against five selected isolates with the highest virulence showed that the interaction between the factors isolates × cultivars was varied and significantly different at P ≤ 0.05 (Figs. 5 and 6).Except for the cv.'Braeburn' which did not produce any symptoms of infection similar to control treatment against all tested fungal isolates, the other cultivars showed symptoms of infection at least against two fungal isolates (Fig. 5).

Discussion
In this study, we found a new species of Cytospora, C. balanejica, associated with crown and collar canker and decline symptoms in young apple trees.The incidence of the disease was greater on the cv.'Red Delicious' than the cv.'Golden Delicious' , indicating higher susceptibility of the first cultivar to this new Cytospora species.Our pathogenicity tests confirmed this, as all studied isolates were pathogenic on the shoots of the cv.'Red Delicious' and had greater virulence (longer necrotic lesions) than the cv.'Golden Delicious' (Fig. 6).Although the disease incidence was significantly lower in the cv.'Golden Delicious' than the cv.'Red Delicious' , it is important to note that the infected plants can provide an inoculum reservoir for the pathogen.
The results of our study showed that the cv.'Braeburn' did not develop any symptoms of infection against all the tested fungal isolates, suggesting that it might have some levels of resistance to the disease.The other 11 examined cultivars showed lesions with various degrees of severity and could be considered susceptible.The resistance of 53 accessions of diverse Malus species and their interspecific hybrids was tested against Valsa ceratosperma (syn.: Cytospora ceratosperma) using excised shoot assay and by measuring the length of necrotic lesion 42 .Fourteen accessions were evaluated as resistant and the highest level of resistance was identified in Malus sieboldii Rehder, which was effective against different isolates of the tested fungus.Similar results were also found in pathogenicity studies using different fungal species and host plants [43][44][45][46][47][48] .The virulence of the tested fungal isolates as measured by lesion length was varied and this could be attributed to the genetic diversity among the isolates.Variability in the lesion length has been reported in pathogenicity evaluations of the isolates of Cytospora spp.and other fungal pathogens 31,46,[49][50][51][52][53] .
Cytospora species generally cause canker, dieback and decline diseases with different symptoms on a wide range of woody perennials including fruit and nut trees, forest and urban trees, and rarely on herbaceous plants with strong ecological adaptability 21,26,41,54,55 .In our study, disease symptoms differed from the previously reported symptoms of apple canker diseases caused by Cytospora species, as the disease starts from the crown and collar region of apple trees (Fig. 1).Other apple diseases such as Neonectria canker, Phytophthora crown, collar and root rots, Rosellinia root rot and fire blight have been reported in the literature to cause similar symptoms on affected young apple trees 13,56 .The similarity in symptoms caused by C. balanejica and other diseases, especially in the early stages of disease development, makes it difficult to accurately identify the causal agents without further laboratory examination.www.nature.com/scientificreports/Apple is one of the main hosts that suffer severe damage from the Cytospora canker disease 31,57,58 .In a most recent study, eight species of Cytospora were identified from apple trees in Iran 34 , emphasizing the necessity of extensive pathogen surveys in apple production regions.Understanding the exact diversity of pathogenic fungi such as Cytospora spp. is crucial for devising regional management strategies for each species, developing rapid diagnostic tools, screening for resistance, and accomplishing regulatory control measurements 59 .Earlier species www.nature.com/scientificreports/identification in Cytospora has relied on morphological characteristics and host associations; however, these characteristics are not stable and informative, confusing species identification and delimitation 41,55,60 .
The results of our phylogenetic analyses using ITS-rDNA sequences placed C. balanejica together with C. albodisca, C. corylina, and C. olivacea in an unresolved clade, confirmed the poor utility of this genomic region in the differentiation of Cytospora species 21,38,61,62 (Supplementary Fig. S1).Recent studies using a polyphasic approach, morphology, and multi-gene phylogeny, have revealed hidden fungal diversity, and led to the description of several new cryptic Cytospora species 21,23,26,38,41 .Based on our multi-gene phylogenetic analyses, C. balanejica formed a well-defined monophyletic lineage distinct from all other strains with close affinity to C. albodisca and C. corylina, two recently described Cytospora species (Fig. 2).Cytospora albodisca and C. corylina were isolated from the branches of Platycladus orientalis (L.) Franco and Corylus heterophylla Fisch.ex.Trautv. in China showing canker and dieback symptoms, respectively 26,41 .
This study found that apple trees are hosts of a new pathogenic species of Cytospora, which should be considered a potentially important causal agent of apple crown and collar canker disease in the studied area.Because Cytospora species are generally considered wound pathogens, infecting plants through cracks and wounds caused by freezing injuries, leaf scars, sunburn, oil injuries, shade-weakened twigs, and pruning wounds 21,53,63 , more precautions should be taken during grafting.Even though this study extends our knowledge about the role of a new Cytospora species in crown and collar canker disease on young apple trees, more studies are needed to reveal its biology and ecology, assess the susceptibility/resistance of apple cultivars under field conditions, and its host range and epidemiology to the development of effective management strategies.

Collection of samples and fungi isolation
Young apple trees (2-6 years old) showing symptoms of decreased growth, decline, and death from three orchards in Urmia, West Azarbaijan province, Iran, were evaluated.Samples were collected from the crown, collar, and trunk base showing bark and wood discoloration and canker (Fig. 1), placed separately in clean paper bags, and transferred to the laboratory for further investigation.Samples were washed gently under running tap water, then cut into smaller pieces (1 × 1 cm 2 ) from the interfaces of the healthy and diseased tissues, and surface disinfested in 3% sodium hypochlorite solution for 2 min, rinsed again three times with sterile distilled water and blotted dry on autoclave sterilized filter paper.The pieces were plated onto potato-dextrose-agar medium supplemented with streptomycin sulfate and penicillin G (150 ppm each) to inhibit bacterial growth (PDA; Merck, Darmstadt, Germany) in 90 mm diam.glass Petri dishes.Petri dishes were incubated at 25 ± 1 °C in darkness, examined at 24 h intervals and hyphae growing out from the plant tissues were transferred to fresh PDA.Pure cultures were obtained using the hyphal tip method.The purified isolates were maintained on PDA slants containing a piece of filter paper and stored at 4 °C.The isolates were deposited in the Fungal Culture Collection of the Iranian Research Institute of Plant Protection ("IRAN") and the Fungal Culture Collection of Urmia University (FCCUU).

Plant materials
It is noted that plant materials used in this study were legitimate samples from apple orchards and all methods comprising plant studies were performed following the relevant guidelines, regulations, and legislation.Required permission to collect samples of apple trees from various orchards in Urmia, West Azarbaijan province, was obtained.

DNA extraction, PCR amplification, sequencing, and phylogenetic analysis
Total genomic DNA was extracted from the mycelial mass of fungal isolates cultured in potato dextrose broth (PDB, Mirmedia Microbiology, Iran) for 7-10 days using the Exgene™ Cell SV mini kit (GeneAll Biotechnology Co, South Korea) following the manufacturer's instruction.For the preliminary screening of all recovered isolates, www.nature.com/scientificreports/polymorphic banding patterns of the isolates were generated by an ISSR-PCR method with ISSR5 ((GA) 5 YC) primer and were compared.Each polymerase chain reaction (PCR) mixture contained 0.4 μM of the primer, 4 μL of a ready master mix (Taq DNA polymerase 2× Master Mix Red, 2 mM MgCl 2 , Ampliqon Company, Denmark), and about 10 ng of template DNA in a final volume of 10 μL.The thermal cycling condition consisted of an initial denaturation step of 5 min at 95 °C followed by 35 cycles of 45 s at 95 °C, 60 s at 41 °C and 90 s at 72 °C and a final extension step of 10 min at 72 °C.Amplicons were visualized on a 1% agarose gel.Isolates with the same banding pattern were considered as the same taxon.To reveal the phylogenetic relationship among the isolates, three isolates were selected based on ISSR banding pattern and morphological characteristics for multigene sequencing (Table 3).The internal transcribed spacer region of nuclear ribosomal DNA (ITS1-5.8S-ITS2),parts of the nuclear ribosomal large subunit (LSU), translation elongation factor 1-α (tef1-α), RNA polymerase II (rpb2) and actin (act1) genes were amplified using the primer pairs ITS5/ITS4 64 , LR0R/LR7 65 , EF1-728F/ EF-2 66,67 , RPB2-5F2/fRPB2-7cR 68,69 and ACT512F/ACT783R The newly generated sequences were checked and trimmed manually in BioEdit v. 7.2.6 70 and deposited in GenBank (Table 3).Sequences based on the combined dataset (ITS-rDNA, LSU, act1, rpb2, and tef1-α) were aligned using the MAFFT v. 7 online service (https:// mafft.cbrc.jp/ align ment/ server/) 71 for each locus separately by including the sequences of ex-type and representative Cytospora strains available in the literature and adjusted where necessary.The concatenated sequence dataset (ITS-rDNA, LSU, act1, rpb2, and tef1-α) was produced in Mesquite v. 2.74 72 and used for phylogenetic analysis.Multi-gene phylogenetic analyses were done by using Maximum Likelihood (ML), Maximum Parsimony (MP), and Bayesian Inference (BI) methods.ML analysis was conducted in RAxML-HPC BlackBox v. 8.2.12 73 provided by the CIPRES Science Gateway v 3.3 74 .The substitution model was set as GTRGAMMA+I and branch stability was estimated by 1000 bootstrap replications to produce a cladogram with nodal support values.BI was performed in MrBayes v. 3.2.7 75 by using the Markov Chain Monte Carlo (MCMC) method with four chains, 1M generations, and a temperature value of the heated chain of 0.1.Trees were saved every 1000 generations, Burn-in was set to 25%, and posterior probabilities (PP) were determined from the remaining trees.For determining the best-fit evolutionary models required for BI, all individual alignments were evaluated in MrModeltest v2.3 76 using the Akaike Information Criterion (AIC).MP analysis was performed in PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 77 .Trees were inferred using the heuristic search option with 1000 random sequence additions and branch swapping with the tree-bisectionreconnection (TBR) algorithm and gaps were treated as missing data.The bootstrap values with 1000 replicates were performed to determine branch support.Descriptive tree statistics [Tree Length (TL), Consistency Index (CI), Retention Index (RI), and Homoplasy Index (HI)] were calculated for trees generated in the parsimony analysis.Sequences of Diaporthe eres CBS 145040 and Diaporthe vaccinii CBS 160.32 were used as outgroups.The resultant phylogenetic tree was visualized in FigTree v. 1.4.4 78 and edited in graphic design software, Adobe Illustrator CC 2018 (Adobe Inc., San Jose, California).The ultimate concatenated alignment and ML-generated tree file were submitted to TreeBASE (https:// www.treeb ase.org) under the accession number 29113.Sequence data were deposited in the GenBank dataset and their accession numbers are provided in Table 3.

Morphological characterization
Purified cultures were grown on PDA medium, incubated in the dark at 25 ± 1 °C, and examined after three, seven, and 30 days.Radial growth was measured by taking two measurements perpendicular to each other in triplicates 21,57,60,79 .Colony color was determined based on Rayner's color charts 80 .Pycnidia formation was induced on pine needles embedded in 2% water agar [20 g Agar (Merck, Darmstadt, Germany) in 1000 mL distilled water] medium or on one-year-old apple shoots embedded in PDA medium and incubated under near ultraviolet (NUV) light (12 h photoperiod) at room temperature.Both pine needles and apple shoots were autoclave sterilized at 121 °C for 20 min.thrice, with a 24-h interval between each sterilization.Pycnidia formation was checked weekly for 30 days.Hand sections of the conidiomata (both transverse and longitudinal) were prepared and mounted in water or lactic acid and examined for morphological details.Macro-morphological characters including size and arrangement of stromata, presence or absence of conceptacle, number, and diameter of ostioles per ectostromatic disk, arrangement of locules and color, shape, and size of discs were examined using an Olympus SZX-ILLB200 dissecting microscope.Micro-morphological characters including the shape and size of conidia (n = 50) and conidiophores/conidiogenous cells (n = 25) were determined at 1000× magnification under an Olympus AX70 compound microscope with differential interference contrast (DIC) illumination.Adobe Photoshop 2020 v. 2.10.8 software (Adobe Inc., San Jose, California) was used for manual editing.

Pathogenicity trials
Pathogenicity trials were done based on the standard and routine method described in the literature 31,42,44,57,[81][82][83][84]   www.nature.com/scientificreports/Sterile PDA plugs were used as the controls.Six shoots were used for each fungal isolate and control treatment.Inoculated shoots were placed in clean plastic containers containing three layered moistened sterile paper towels and incubated under laboratory conditions (diurnal light, 25 ± 2 °C, 80% relative humidity) for 21 days.All the experiments were repeated once under similar conditions.Length of bark and wood discoloration around the inoculated sites were measured 21 days post-inoculation.Also, the pathogenicity of the most virulent isolate (BA 2-4) was determined on the 'Red Delicious' cultivar under field conditions.Four 2-3-year-old branches in four geographical directions were selected.The bark of the branches was surface disinfected by spraying with 75% ethanol, and the fungal inoculation was the same as described for detached shoots.Inoculation was done on April 7, 2023, and the results were evaluated on May 24, 2023.
In addition, five fungal isolates (BA 1-1, BA 2-1, BA 2-4, KU 1-1, and BA 3-1 isolates) which had the highest virulence in the trials as mentioned above (Table 1) were chosen for the evaluation of reaction of 12 apple cultivars including ʻBraeburn' , ʻDelbard Estivale' , ʻFuji' , ʻGranny Smith' , ʻGolden Delicious' , ʻGolden Primrose' , ʻIdared' , ʻRed Delicious' , ʻM4' , ʻM7' , ʻMM106' and ʻMM109' against these isolates.Healthy shoots were collected from the apple cultivar collection farm of Urmia University and used for pathogenicity tests as described above and the length of bark and wood discoloration around the inoculated sites was measured 21 days postinoculation.Experiments were laid down following a completely randomized design (CRD).The pathogenicity data were transformed by square root due to the existence of zero values and were subjected to analysis of variance (ANOVA) using SAS v.9.1 software (SAS Institute, Inc., USA).The lesion length means were compared with Duncan's multiple range test (P ≤ 0.05).To confirm Koch's postulates, fungal re-isolation was carried out from the margins of the developed lesions in all symptomatic samples, and isolates were re-identified morphologically as described previously.

Figure 1 .
Figure 1.Typical symptoms of crown and collar canker and necrosis on naturally infected young apple trees cvs.'Red Delicious' and 'Golden Delicious' .(a, b) cv.'Red Delicious' .(c) cv.'Golden Delicious' .(d-i) Disease symptoms on the crown, collar and trunk of the cvs.'Red Delicious' and (j) 'Golden Delicious' .(k-m) Cross sections showing disease progress in the infected trunks of the cv.'Red Delicious' .

Figure 2 .
Figure 2. Maximum Likelihood (ML) tree based on combined ITS, LSU, rpb2, act1 and tef 1-a sequences matrix in different Cytospora species.The Maximum Likelihood, Maximum Parsimony (MP) bootstrap support values and posterior probabilities of Bayesian inference (BIPP) > 50% are given at the nodes (ML/MP/BI).The tree was rooted to Diaporthe eres CBS 145040 and Diaporthe vaccinii CBS 160.32.The scale bar indicates the number of nucleotide substitutions.

Figure 4 .
Figure 4. Pathogenicity tests of the most virulent isolate (BA 2-4) on apple cv.'Red Delicious' under field conditions.(A-D) Inoculation process.(E, F) Bark and wood discoloration and necrosis 45 days post inoculation.

Figure 6 .
Figure 6.Mean lesion lengths (mm) of 12 apple cultivars inoculated with five selected isolates of Cytospora balanejica based on Duncan's multiple range test.Different letters show statistically significant differences at P ≤ 0.05.

Table 2 .
Phylogenetic information of individual and combined sequence datasets used in phylogenetic analyses.a Akaike Information Criterion Substitution models implemented in Bayesian Inference.
Vol.:(0123456789) Scientific Reports | (2024) 14:6629 | https://doi.org/10.1038/s41598-024-57235-3 67, respectively.All primers were purchased from Pishgam Company, Tehran, Iran.The PCR mixtures for all reactions consisted of about 10 ng/µL of genomic DNA, 0.4 µM of each primer, and 12.5 µL of 2× ready-to-use reaction mix (Taq DNA polymerase 2× Master Mix Red, 2 mM MgCl 2 , Ampliqon, Denmark) in a total volume of 25 µL.Thermal conditions for PCR amplification of ITS, LSU, act1, and tef1-α consisted of an initial denaturation step of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C and 60 s at 72 °C, and a final extension step of 5 min at 72 °C.The part of the rpb2 gene was amplified using touch-down PCR consisting of an initial denaturation step of 5 min at 95 °C, followed by 40 cycles of 45 s at 95 °C, 45 s at 60-55 °C (annealing temperature decreased 0.5 °C in the first 10 cycles), 45 s at 72 °C and a final extension step of 10 min at 72 °C.PCR products were visualized on a 1.5% Agarose gel (100 V for 30 min) stained with CyberSafe (Safe DNA Stain, 6X Pishgam, Iran), following the manufacturer's instruction to confirm the amplicon presence and size.Amplification products were purified and sequenced by Macrogen Inc. (Seoul, South Korea).

Table 3 .
Fungal isolates used in the molecular analyses in this study and GenBank accession numbers.a ATCC: American Type Culture Collection, Virginia, USA; CBS: Westerdijk Fungal Biodiversity Institute (CBS-KNAW Fungal Biodiversity Centre), Utrecht, The Netherlands; CFCC: China Forestry Culture Collection Centre, Beijing, China; CMW: Culture collection of Michael Wingfield, University of Pretoria, South Africa; CPC: Culture collection of Pedro Crous, The Netherlands; IMI: Culture collection of the International Mycological Institute, CABI Bioscience, Egham, Surrey, UK; MFLU: Mae Fah Luang University herbarium, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Thailand; MUCC: Murdoch University Culture Collection, Perth, Australia; NE: Gerard Adams collections, University of Nebraska, Lincoln NE, USA; XJAU: Xinjiang Agricultural University, Xinjiang, China; IRAN: the Fungal Culture Collection of the Iranian Research Institute of Plant Protection; FCCUU: Fungal Culture Collection of Urmia University.NA: not applicable.All the new isolates used in this study are in bold and the type materials are marked with T .